Lipoproteins are water soluble conjugated proteins. They are conjugated with lipids-substances such as cholesterol, triglycerides and phospholipids. Cholesterol, triglycerides and phospholipids are all insoluble in aqueous solutions. They are conjugated to proteins which make them soluble in serum.
The association of cholesterol and lipoproteins with arteriosclerosis has led to the frequent evaluation of cholesterol and lipoprotein in human serum. It is known that elevated total cholesterol and elevated low density lipoprotein (LDL) cholesterol levels may lead to poor cardiovascular health while elevated high density lipoprotein (HDL) cholesterol does not have a negative influence on cardiovascular health and may even improve the condition of cardiovascular health. Because of the association of cholesterol and lipoproteins with cardiovascular health and because it has been demonstrated that LDL cholesterol can be decreased and HDL cholesterol increased by diet, exercise and drugs, several clinical assays have been developed to measure lipoprotein, triglycerides and cholesterol.
A typical assay for lipid analysis often includes values for cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, phospholipids, cholesterol esters, free fatty acids, apolipoprotein A1 and apolipoprotein B. In routine practice, LDL cholesterol is estimated using the Friedwald equation wherein LDL cholesterol is calculated by subtracting measured HDL cholesterol and estimated very low density lipoprotein (VLDL) cholesterol (approximated as one-fifth of the serum triglyceride) from total cholesterol. HDL cholesterol may be measured by various methods such as density separation using ultracentrifugation, electrophoresis or enzyme-linked methods employing cholesterol oxidase and photometric measurement. When the later method is employed, the VLDL. and LDL cholesterol is first precipitated from the serum with reagents such as heparin, dextran sulfate and magnesium ion or phosphotungstate. The HDL cholesterol remaining in the supernatant can then be measured.
Controls are used to objectively evaluate the accuracy and precision of lipoprotein testing procedures. Unfortunately, the freezing and lyophilization of lipoprotein controls leads to aggregation and turbidity of lipoprotein solutions. Another disadvantage in lipoprotein controls is reconstitution of lyophilized controls which can lead to inaccuracies due to volumetric measurements. Various approaches to improving lipid controls (triglycerides and total cholesterol) have thus been developed.
For example, in U.S. Pat. Nos. 4,816,411 and 4,503,146 (incorporated by reference), turbidity in biological fluid such as plasma or serum is reduced by adding a surfactant and cholesterol esterase or lipase, to degrade the lipoproteins. These patents describe photometric assays for cholesterol and triglycerides, wherein clear samples are essential. U.S. Pat. Nos. 4,626,511 and 5,310,679 (incorporated by reference), also disclose reduction of turbidity by the addition of a surfactant and lipolytic enzyme to degrade the lipoproteins.
In U.S. Pat. No. 4,011,045 (incorporated by reference), lipoproteins are precipitated from serum by the addition of divalent cations and dextran sulfate. The large lipoproteins are discarded and the short chain triglycerides are added back to the serum. The added triglycerides in serum are then emulsified by using the surfactant alkylphenoxypolyethoxyethanol. The serum is then lyophilized and reconstituted to produce a clear preparation.
Similarly, in U.S. Pat. No. 3,955,925 (incorporated by reference), a clear, lyophilized control product is prepared by precipitating lipoproteins from serum with dextran sulfate and divalent cations and adding back to the serum bovine high density lipoprotein which has been purified. Surfactant is also used to remove turbidity in U.S. Pat. Nos. 5,258,315, 4,579,825 and 4,708,939 (incorporated by reference), although no control product is disclosed. Similarly in U.S. Pat. No. 4,289,649 (incorporated by reference), non-ionic and ionic detergent is used to remove turbidity in a control for triglycerides and total cholesterol.
In U.S. Pat. No. 4,127,502 (incorporated by reference), the addition of arabinose, sorbitol, sucrose or glucosamine was shown to reduce the turbidity of lyophilized serum and U.S. Pat. Nos. 4,298,441, 4,045,176 and 4,216,117 (incorporated by reference), disclose that lyophilized control serum which contains non-reducing sugars is stable.
The following U.S. Patents and journal article, which are incorporated by reference herein, relate generally to lipid preparations: U.S. Pat. No. 3,751,381, 3,853,465, 4,189,400, 4,290,774, 3,260,648, 4,414,326 and Hirst, C. F. et al., J. Clin. Pathol. 45:701-703 (1992).
None of the above patents describe stable lipoprotein controls; they are all used for cholesterol and triglyceride standards only. Several recent journal articles make it clear that there is no lipoprotein control which adequately behaves like human serum. In particular, Naito et al., Arch. Pathol. Lab. Med. 117:345-351 (1993), describes the "matrix effects" of known lipoprotein controls used in proficiency studies. Matrix is defined by the National Committee for Clinical Laboratory Standards, Document EP10-T, Vol. 9, No. 3, November, 1988, as "all properties other than the property to be measured, that can have an effect on a measured value," and matrix effects is defined as "an analytic bias due to the matrix of the processed specimen being measured, that is exclusive of calibration." Naito points out that the matrix problems associated with lipoprotein controls severely hamper interlaboratory accuracy, standardization efforts, and monitoring of laboratory performance. It is thus suggested that efforts be made to improve lipoprotein controls so they behave more like human serum.
Likewise, in Kroll, M. H. et al., Clin. Chem. 40:389-394 (1994), it was shown that there is a lack of linearity in high density lipoprotein measurements. Available commercial controls were not linear and it was suggested that the non-linearity is due to alteration of the lipoproteins during lyophilization. Rej. R., Clin. Chem. 40:345-346 (1994) also addresses the inadequacy of available lipoprotein controls and stresses the importance of developing adequate preparations for standardization of methods. The level of importance of this issue is further indicated by an Omnibus Solicitation of the National Institutes of Health, PHS 95-1 (Dec. 1, 1994 Application Receipt Date). The title of the solicitation is "Development of proficiency testing materials free of the matrix effects that compromise accurate measurement of total cholesterol, HDL cholesterol, and LDL cholesterol."
It would thus be desirable to provide an accurate, stable, lipoprotein control. It would further be desirable to provide an accurate, stable, liquid, lipoprotein control that is not lyophilized and reconstituted or frozen and thawed. It would also be desirable to provide an accurate, complete, chemistry control which includes all lipoproteins.